Research

Overview/Introduction

Our immune system protects us against diseases by identifying and fighting pathogens and tumor cells. It is able to distinguish between pathogens, such as viruses, bacteria or parasitic worms, and the organism?s own healthy cells and tissues.

T cells belong to the group of white blood cells and play a central role in the adaptive immune system. T cell mediated immunity is essential for a proper protection against infection and malignant diseases.

In the course of organ transplantation chronic immune suppression is necessary to avoid organ rejection. Thus, an efficient T cell mediated immunity against pathogens, such as a virus, is inhibited by immunosuppressive drugs.

In this case, adoptive cellular therapy with virus-specific T cells has proven to be an efficient and beneficial treatment option in the clinics. Here, virus-specific T cells from healthy individuals are adoptively transferred into patients that received a new organ and immunosuppressive drugs. However, such virus-specific T cells can only be isolated from virus sero-positive individuals. Thus, the need of new technologies allowing a direct isolation of virus-specific T cells is demanding.

Furthermore, in organ transplantation frequently virus sero-positive donor organs are transplanted into virus sero-negative recipients. Particularly children are at high risk. Therefore efficient methods enabling a direct analysis and isolation of antigen-specific na?ve T cells is necessary.

The aim of our project is to selectively isolate antigen-specific T cells according to activation marker signatures using novel methodologies. We anticipate that the new approaches will prove as serious candidates for application in clinical treatment of infectious and malignant diseases.

The individual projects organized by the participating scientists and medical doctors provide an excellent prerequisite for translating efficient and optimal technologies into clinical applications.

Project 1: High efficiency T cell in vitro priming (T-PRIME)

The adoptive T cell therapy is well working for fighting viral diseases if cells are generated from sero-positive donors/patients. However, there is strong medical need to manage severe viral infections in na?ve sero-negative patients.

For sero-negative patients T lymphocytes need to be induced and expanded in vitro requiring repeated stimulation of na?ve precursors with professional antigen-presenting cells. To circumvent the laborious cell culture periods we want to employ the direct isolation of antigen-specifically activated na?ve T cells according to specific activation marker signatures. After high-gradient magnetic cell sorting (MACS) that enables the isolation of rare frequencies of antigen-specifically activated na?ve precursors, a simplified cell culture expansion can be performed.

We aim to employ new strategies based on the isolation of specific T cells according to their activation markers independent of their functional differentiation status of the activated T cell.

Project 2: Activation marker signatures (A-SIGNAT)

Immune cells can be specifically isolated by their surface marker expression. A widely used technique is magnetic activated cells sorting (MACS) to separate cells according to their surface markers.

For our study we use two surface markers (CD154 and CD137) to specifically and efficiently isolate antigen-specific na?ve T cells.

Aim of this project is to adapt these procedures established only for research into clinical applications. As a result we will establish clinical-grade compatible procedures including the isolation of antigen-specific T cells from primary short-term in vitro cultures in a GMP-grade manner (Good manufacturing practice).

One major drawback of MACS is that cell separation procedures (particularly those applicable for clinical use) can only be applied for single markers expressed by target cells. Therefore, we aim to enhance the sensitivity of the magnetic cell sorting procedure by establishing a procedure that enables the isolation of cells co-expressing two cell surface markers of interest. This increases the sensitivity of detection and also specificity of isolating any rare cell population of interest.

Project 3: Functional & molecular analysis (T-F UNCTION)

Subproject 3 aims to evaluate the functional potential of the in vitro generated and isolated T cell populations on the molecular level and in established pre-clinical models.

On the one hand we will use molecular technologies such as gene expression and particularly epigenetic profiling analysis. This offers a great potential to evaluate the functional potential of T cells.

Measuring DNA methylation of distinct gene regions and specific gene expression profiles in standardized assays using few sample material and don?t need of antigenic in vitro restimulation can replace laborious functional assays. Thus, we anticipate the development of new clinical applicable diagnostic procedures that can be performed as quality tests for adoptive T cell from donors.

Additionally, we will use in vivo animal models to check the efficacy/safety of T cell transplants before going into clinical trials.

Project 4: GMP-reagent and processing (T-REAGENT)

The principal investigator of this subproject is Miltenyi Biotec, a leading company on the world market providing tools for cellular therapy. Since more than ten years Miltenyi is developing innovative GMP-compliant isolation technologies, culture systems and cellular analysis tools for T cells and various other leukocytes.

Current studies are focusing on the isolation and expansion of antigen-specific T cells, such as CMV-, EBV-specific T cells for the treatment of viral infections in the context of hematopoietic stem cell therapy.
In the S-T-THERA project we aim at the translation of existing research protocols into clinical procedures for the generation of T cell products. This comprises on the one hand the development of appropriate antigen preparations and cell culture tools for the envisaged in vitro T cell stimulations. On the other hand, GMP-grade reagents for magnetic cell sorting like suitable antibodies specific for CD154 and CD137 will be developed. These tools will be used to establish and validate a final protocol for the generation of antigen-specific T cells for the envisaged clinical trial (see Project 2).

Project 5: Preparation/design of clinical study (S-T-THERA-STUDY)

The final project intends to design and organize a clinical study using the results obtained by Project 1-4. The principal investigator of this project has already been leading several investigator-driven and company-sponsored clinical phase II/III trials in transplant patients.

Leading the immune therapy platform at the BCRT and the Interdisciplinary Transplantation Immunology Group she has designed and conducted several clinical pilot and phase II trials aiming to treat viral infections particularly in the course of organ transplantation.

Furthermore, a strong expertise in preparing all necessary relevant documents is established in the BCRT clinical study management group involved in project 5.

This project aims to develop the first project for a clinical trial with virus-specific T cells in sero-negative patients. There is a high medical need for an adequate prophylactic/preemptive antiviral therapy in sero-negative transplant patients (particularly if getting a graft from sero-positive donor) as prophylactic use of antiviral drugs is not (EBV) or partly (CMV) effective only, expansive, and harbor the risk of drug resistance development.